Isoprenoids are a family of compounds derived from an isoprene building block. Isoprenoids include primary metabolites such as sterols, carotenoids, growth regulators, and the polyprenol substituents of dolichols, quinones, and proteins. These compounds are essential for membrane integrity, photoprotection, orchestration of developmental programs, and anchoring essential biochemical functions to specific membrane systems, respectively. Isoprenoids also include secondary metabolites such as monoterpenes, sesquiterpenes, and diterpenes.
Isoprenoids are made via the isoprenoid biosynthetic pathway. The common denominator for this diverse array of compounds is a universal five-carbon building block, isoprene. The polymerization of two diphosphorylated isoprene building blocks (e.g., isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP)) generates geranyl diphosphate (GPP), a linear C10 intermediate that can be converted to cyclic or linear end-products representing the monoterpenes, or used in another round of polymerization. The addition of a third isoprene unit to GPP generates which can also be converted to cyclic or linear products representing the sesquiterpene class. Continuing the polymerization and chemical differentiation cycle leads to the production of other classes of terpenoids named according to the number of isoprene building blocks leading to their biosynthesis, for example, the addition of a third IPP to FPP generates geranylgeranyl diphosphate (GGPP).
These polymerization reactions are catalyzed by prenyl transferases that direct the attack of a carbocation (an electron deficient carbon atom resulting from the loss of the diphosphate moiety of one substrate) to an electron-rich carbon atom of the double bond on the IPP molecule. The enzymes responsible for the cyclization of GPP, FPP, and GGPP are referred to as monoterpene, sesquiterpene, and diterpene synthases or collectively, terpene synthases, and represent reactions committing carbon from the general isoprenoid pathway to end products in the monoterpene, sesquiterpene, and diterpene classes, respectively.
An important biochemical distinction between the prenyl transferase and isoprenoid synthase reactions are that the prenyl transferases catalyze carbon-carbon bond formation between two substrate molecules, whereas the synthases catalyze an intramolecular carbon-carbon bond formation.
Increasing the activity of a prenyl transferase and/or an isoprenoid synthase in a cell can increase the terpene and/or terpenoid content of the cell. In addition, if a cell contains a terpene and/or terpenoid that mediates a desirable phenotype (for instance, a biocidal compound that confers a crop protection phenotype), increasing the activity of the appropriate prenyl transferase and/or isoprenoid synthase may increase accumulation of the compound in the cell.
Described herein are modified prenyl transferase and isoprenoid synthase enzymes useful in the production of terpenes and terpenoids. Also described herein are methods for the production of terpenes and terpenoids.